# Synthesis and Biological Activity Characterization of Vascular Endothelial Growth Factor Using an Optimized Wheat Germ Cell-Free System

## TL;DR

Researchers developed a cell-free system to efficiently produce functional human vascular endothelial growth factor, which promotes cell growth and blood vessel formation.

## Contribution

An optimized wheat germ cell-free system with a self-renewing ATP source for producing bioactive eukaryotic proteins.

## Key findings

- The engineered E. coli strain produced ATP efficiently using a three-enzyme cascade.
- The cell-free system successfully synthesized active human VEGF165.
- VEGF165 induced strong angiogenic responses and cell migration in vitro.

## Abstract

Cell-free protein synthesis has become a powerful tool for producing functional proteins, circumventing many limitations of live-cell systems. Platforms based on wheat germ extract are favored for their high efficiency in translating and folding complex eukaryotic proteins. To overcome the energy limitation common in such systems, we engineered an Escherichia coli strain to function as a self-renewing ATP source. This strain co-expresses a three-enzyme cascade—adenosine kinase, adenylate kinase, and acetate kinase—that efficiently converts adenosine and acetyl phosphate into ATP. Using the lysate from this biocatalyst to energize an optimized wheat germ extract, we established a high-performance cell-free synthesis platform. This integrated system supported the robust production of multiple recombinant proteins. As a key demonstration, we synthesized human vascular endothelial growth factor 165, which exhibited full biological activity. The cell-free-produced VEGF165 significantly stimulated the proliferation of human umbilical vein endothelial cells (HUVECs) and human skin fibroblasts (HSFs). It also potently induced angiogenic responses, including the formation of extensive, interconnected capillary-like networks by HUVECs in vitro and accelerated cell migration in scratch-wound assays. Our work establishes a scalable and efficient platform for on-demand production of bioactive eukaryotic proteins, highlighting its considerable potential for advancing regenerative medicine and related therapeutic applications.